Retroviruses integrate a DNA copy of their genome into host DNA as an obligatory step in their replication cycle. Our work focuses on the molecular mechanism of integration, and in particular on the structure and function of HIV integrase. Integrase is the viral enzyme that carries out the key DNA cutting and joining steps in the integration reaction. The structures of each of the individual domains of HIV-1 integrase have been determined, but their spatial arrangement in the active complex with DNA substrate is unknown. All structures of the catalytic domain of HIV-1 integrase determined to date are in the absence of DNA. However, the presence of DNA in the active site has been shown to strongly influence the binding of inhibitors. It is therefore likely that high-resolution structures of inhibitors bound to an active site engaged with DNA substrate will be required to understand their binding in detail and serve as the basis for the design of better derivatives. A major unresolved problem is the organization of integrase with DNA substrate in the active nucleoprotein complex. Based on the crystal structure of the catalytic together with the N-terminal domain of HIV integrase, the crystal structure of the closely related Tn5 transposase in complex with DNA, and cross-linking data we have developed a tentative model of the path of viral DNA on the integrase surface in the vicinity of the active site. By introducing cysteines into the integrase and SH groups at various positions in the DNA substrate we are attempting to generate complexes that retain 3? processing activity with the DNA substrate covalently attached to the protein; such complexes would partially map the DNA path in the active complex and would be useful for crystallization trials. The major impediment to further structural, biophysical and further biochemical studies of retroviral DNA integration is that it has not yet been possible to reconstitute a nucleoprotein complex from purified integrase and DNA substrate that exhibits all the properties of complexes isolated from infected cells. In particular, the complexes are not competent for efficient two-ended integration and aggregate under reaction conditions. We have studied conditions for optimizing two-ended integration in order to obtain nucleoprotein complexes with properties that more faithfully reproduce those of complexes isolated from infected cells. Although integrase is the key enzyme in the integration process, integrase does not function in isolation in vivo. Rather, it forms part of a large nucleoprotein complex, the preintegration complex, derived from the core of the infecting virion. We are studying Moloney murine leukemia virus preintegration complexes to elucidate the role of other viral and cellular proteins in retroviral DNA integration. In order to successfully integrate into the host genome, retroviruses must avoid self-destructive integration into their own DNA (autointegration). We have identified a cellular protein (BAF) that blocks self-destructive autointegration of retroviral DNA. BAF is non-specific DNA binding protein that is highly conserved among multicellular eukaryotes. The molecular mechanism by which BAF blocks autointegration has been determined. The cellular function of BAF, and its interacting partners, is currently under investigation together with the potential role of these other cellular proteins in the retroviral replication cycle.